Cholesterol and ergosterol superlattices in three-component liquid crystalline lipid bilayers as revealed by dehydroergosterol fluorescence

We have examined the fractional sterol concentration dependence of dehydroergosterol (DHE) fluorescence in DHE/cholesterol/dimyristoyl-L-alpha-phosphatidylcholine (DMPC), DHE/ergosterol/DMPC and DHE/cholesterol/dipalmitoyl-L-alpha-phosphatidylcholine (DPPC) liquid-crystalline bilayers. Fluorescence intensity and lifetime exhibit local minima (dips) whenever the total sterol mole fraction, irrespective of the DHE content, is near the critical mole fractions predicted for sterols being regularly distributed in hexagonal superlattices. This result provides evidence that all three of these naturally occurring sterols (e.g., cholesterol, ergosterol, and DHE) can be regularly distributed in the membrane and that the bulky tetracyclic ring of the sterols is the cause of regular distribution. Moreover, at the critical sterol mole fractions, the steady-state anisotropy of DHE fluorescence and the calculated rotational relaxation times exhibit distinct peaks, suggesting that membrane free volume reaches a local minimum at critical sterol mole fractions. This, combined with the well-known sterol condensing effect on lipid acyl chains, provides a new understanding of how variations in membrane sterol content change membrane free volume. In addition to the fluorescence dips/peaks corresponding to hexagonal superlattices, we have observed intermediate fluorescence dips/peaks at concentrations predicted by the centered rectangular superlattice model. However, the 22.2 mol% dip for centered rectangular superlattices in DHE/ergosterol/DMPC mixtures becomes diminished after long incubation (4 weeks), whereas on the same time frame the 22.2 mol% dip in DHE/cholesterol/DMPC mixtures remains discernible, suggesting that although all three of these sterols can be regularly distributed, subtle differences in sterol structure cause changes in lateral sterol organization in the membrane.     

Change in the composition of Candida albicans cells during development of resistance to polyene antibiotics

The composition of lipid and protein components of the membrane structures was studied in the strains of Candida albicans susceptible and resistant to polyene antibiotics. The content of sterols was lower and the content of free fatty acids was higher in the cells of resistant strains cf. susceptible strains. The composition of sterols was also different in the resistant strains: analysis of absorption spectra of sterol extracts in heptane in UV revealed complete absence of ergosterol, the main component of the susceptible strain, and appearance of a heterogenous peak at 215--240 nm.     

Molecular organization and dynamics of 1-palmitoyl-2-oleoylphosphatidylcholine bilayers containing a transmembrane alpha-helical peptide

The molecular organization and dynamics have been investigated in membranes consisting of 1-palmitoyl-2-oleoyl-l-alpha-phosphatidylcholine (POPC) and various ratios of a transmembrane alpha-helical peptide, Ac-K2L24K2-amide (L24), in order to gain insights into how the transmembrane portions of membrane proteins are mixed with phospholipids and organized in biological membranes. Particular attention was paid to membranes with high peptide concentrations. The molecular organization and dynamics were studied in the ps-to-micros regime using various spin-labeling techniques. Conventional ESR spectra as well as saturation-recovery curves measured in both the presence and the absence of molecular oxygen showed that phosphatidylcholine spin-labels detect the existence of a single homogeneous environment, indicating that both L24 and POPC are likely to be undergoing fast translational diffusion in L24-POPC membranes of up to 9 mol % peptide. Since 16-18 molecules of phosphatidylcholine are required to surround a transmembrane alpha-helical peptide [Morrow, M. R., Huschilt, J. C., and Davis, J. H. (1985) Biochemistry 24, 5396-5406], L24 must form L24-rich regions at a P/L ratio of 1/10 instantaneously. However, these results suggest that the lipid exchange rates among the bulk, boundary, and L24-rich regions are fast, and that the L24-rich regions must be forming and dispersing rapidly in a time scale shorter than 0.1 micros, the conventional ESR spin-label time scale and the electron spin-lattice relaxation time scale in the presence of molecular oxygen. Although this does not exclude the possibility of the formation of small, stable oligomers of L24, it is unlikely because L24 lacks features that would favor their formation. L24 (9 mol %) increases the hydrophobicity of the central part of the POPC membrane from the level of 1-decanol to that of pure hexane and also increases the hydrophobicity near the membrane surface from the level of 2-propanol to that of 1-decanol. The effect of 9 mol % L24 on the order parameter profile is similar to that of decreasing the temperature by approximately 8 degrees C between 10 and 55 degrees C. It is concluded that L24 is highly miscible in POPC membranes even at high concentrations in the membrane.     

Sphingomyelinase induces lipid microdomain formation in a fluid phosphatidylcholine/sphingomyelin membrane

The behaviors of two chemically well-defined sphingolipids, N-palmitoyl-sphingomyelin (C16:0-SM) and the corresponding ceramide (C16:0-Cer), in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) matrix were compared. Minor attenuation of lateral diffusion upon increasing the mole fraction of C16:0-SM (XSM, up to 0.25) was indicated by the slight decrement in the excimer/monomer intensity ratio (Ie/Im) for a trace amount (mole fraction X = 0.01) of a pyrene-labeled ceramide analogue (N-[(pyren)-1-yl]decanoyl-sphingosine, PDCer) in keeping with the miscibility of C16:0-SM in POPC. Increasing membrane order was revealed by the augmented polarization P for diphenylhexatriene (DPH). In contrast, when C16:0-Cer was substituted for C16:0-SM an approximately 1.6-fold increase in Ie/Im for PDCer was evident upon increasing Xcer, with parallel increment in DPH polarization. In agreement with our recent data on natural ceramides in dimyristoylphosphatidylcholine (DMPC) bilayers [Holopainen et al. (1997) Chem. Phys. Lipids 88, 1-13], we conclude that C16:0-Cer becomes enriched into microdomains in the fluid POPC membrane. Interestingly, enhanced formation of microdomains by ceramide was observed when the total sphingolipid content in tertiary alloys with POPC was maintained constant (Xcer + XSM = 0.25) and the SM/Cer stoichiometry was varied. Finally, when ceramide was generated enzymatically in POPC/C16:0-SM (3:1, molar fraction) LUVs by sphingomyelinase (SMase, Bacillus cereus), maximally approximately 85% of hydrolysis of sphingomyelin was measured within <3 min at 30 degreesC. The formation of ceramide was accompanied by a closely parallel increase in DPH polarization. There was also an increase in Ie/Im for PDCer; however, these changes in Ie/Im were significantly slower, requiring approximately 105 min to reach a steady state. These data show that the rapid enzymatic formation of ceramide under these conditions is followed by much slower reorganization process, resulting in the formation of microdomains enriched in this lipid.     

Possible nystatin-protein interaction in yeast plasma membrane vesicles in the presence of ergosterol. A F?rster energy transfer study

The F?rster energy transfer from tryptophan residues of membrane proteins to nystatin was measured in reconstituted yeast plasma membrane vesicles free of, or doped with, ergosterol. We wanted to elucidate whether the functional change of membrane transport proteins from H+ symporters to facilitators, observed in ergosterol-containing plasma membrane vesicles on addition of nystatin [Opekarová and Tanner (1994) FEBS Lett. 350, 46-50], is reflected in altered protein-nystatin relations within the membrane. Both frequency-domain and time-domain time-resolved fluorescence spectroscopy showed that in the presence of ergosterol nystatin is located much closer to membrane proteins than in its absence.     

Effect of cholesteryl hemisuccinate on the interfacial properties of phosphatidylcholine bilayers

Cholesteryl hemisuccinate (CHEMS) is an amphipathic lipid that can regulate cell growth. A comparison of the effects of CHEMS and cholesterol on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers was investigated using fluorescence techniques. In liquid-crystalline phase POPC bilayers, CHEMS increased the interfacial surface charge, but was less effective than cholesterol in reducing acyl chain mobility and interfacial hydration. In liquid-crystalline phase DPPC bilayers, CHEMS and cholesterol were equally effective in reducing acyl chain mobility. Similar to the POPC matrix, CHEMS increased the interfacial surface charge and cholesterol decreased the surface hydration. The different effect of cholesterol and CHEMS on acyl chain mobility may be due to a preferential interaction of cholesterol with POPC. In gel phase DPPC bilayers, CHEMS and a succinylated pyrenyl cholesterol analog exhibited different effects on membrane physical-chemical properties than cholesterol. Succinylation also increased the rate of transfer of the pyrenyl cholesterol analog between single unilamellar vesicles approximately seven fold. This process demonstrated first-order kinetics which indicated that transbilayer migration was not a rate-limiting step. The succinylation of cholesterol places a carboxyl group at the lipid-water interface and the sterol ring deeper in the bilayer. For a structural model to explain its biological properties, CHEMS should be considered a bulky fatty acid.     

Physical connections between feeder cells and recipient normal mammary epithelial cells

Amphotericin B (AmB) is the most widely used polyene antibiotic to treat systemic fungal infections which affect an increasing number of immunocompromised patients. It is generally thought that AmB forms pores within the fungi membranes by interacting with ergosterol, the main sterol of fungi. However, it also interacts with the cholesterol contained in mammalian cells, hence its toxicity. In order to have a better understanding of the interactions prevailing between AmB and sterols, differential scanning calorimetry was used to study various mixtures incorporating from 6.5 to 25 mol% of AmB in pure dipalmitoylphosphatidylcholine (DPPC) vesicles and in ergosterol- or cholesterol-containing DPPC vesicles. The sterol concentration was kept constant at 12.5 mol% with respect to the phospholipid. Our results show that three phases co-exist when AmB is dispersed in the pure phospholipid. One corresponds to the phospholipid phase alone. The two others are characterised by a broad transition at temperatures higher than the main transition temperature of the pure phospholipid, corresponding to the drug in interaction with the aliphatic chains of the lipid. The fact that the transition temperatures of these additional components are higher than that of the pure phospholipid suggests that AmB interacts strongly with the aliphatic chains of the lipid, consistent with the idea prevailing in the literature that AmB by itself may form pores in a lipid matrix. When AmB interacts with cholesterol-containing bilayers the thermograms also present three components. Upon increasing the concentration of AmB, though, an important broadening of these components is observed which is explained in terms of destabilisation of the organisation of the aliphatic chains. The situation is strikingly different if ergosterol is present in the lipid matrix. The thermograms remain unmodified as the concentration of AmB is increased and a broad transition, now involving only two components when the thermograms are decomposed, is observed. An analysis of the results shows that various interacting units, e.g. AmB+DPPC and (AmB+ergosterol)+DPPC, are present within the membrane. These units involve the phospholipid and hence contribute to its structurisation. The important differences between the thermograms obtained with the ergosterol- as compared to the cholesterol-containing bilayers, in spite of the structural similarity of these two sterols, provides strong evidence for the selectivity of interaction of AmB with ergosterol as compared to cholesterol. It is thus clear that the action of AmB on cholesterol- as compared to ergosterol-containing membranes results from different mechanisms. Finally, UV-visible spectra of AmB in pure as well as sterol-containing DPPC vesicles show the presence of absorption bands that give support to the interpretation derived from the calorimetric data.     

The interaction of the polyene antibiotic lucensomycin with cholesterol in erythrocyte membranes and in model systems. III. Characterization of spectral parameters

The variations of optical density and fluorescence of lucensomycin are good indices of the binding of this polyenic antibiotic to membranes. The former parameter reflects more generally the binding to any site present in the membrane, while the latter is more specific for binding to cholesterol. The chromophore of the lucensomycin-cholesterol complex has a relatively long lifetime, is almost immobile in the membrane, and is not accessible to water-soluble fluorescence-quenching agents. The stoichiometry, evaluated fluorometrically, corresponds to about two cholesterol molecules per polyene. In colloidal cholesterol suspensions, the extent of binding as a function of free polyene concentration is described by rectangular hyperbolae, the dissociation constant being, however, dependent on the sterol concentration. In erythrocyte membranes, on the other hand, and even more markedly in model systems containing appropriate solvents, the combination between lucensomycin and the sterol sites is described by sigmoid titration curves, indicative of cooperative effects, and probably due to solvation of cholesterol.     

Nystatin pastilles and suspension in the treatment of oral candidosis

Clinical audit revealed that the treatment of oral candidosis was more successful with nystatin pastilles than with nystatin suspension. The purpose of this investigation was to determine the reasons for this observation. The concentration of nystatin needed to kill 49 consecutive clinical isolates of Candida albicans was measured. The isolates varied in cidal concentrations from 1.875 to 30 U/ml. The time taken to kill these isolates at their cidal concentrations was found to vary from 120 to 300 min. Volunteer studies showed that antifungal activity in the oral cavity was eliminated rapidly after the use of nystatin suspension. In contrast, the polyene could be detected for at least 5 hours after use of the nystatin pastille. The nystatin pastille can be expected to be more effective at killing Candida albicans than the suspension due to its persistent effects.     

Itraconazole resistance in Aspergillus fumigatus

Invasive aspergillosis is an increasingly frequent opportunistic infection in immunocompromised patients. Only two agents, amphotericin B and itraconazole, are licensed for therapy. Itraconazole acts through inhibition of a P-450 enzyme undertaking sterol 14alpha demethylation. In vitro resistance in Aspergillus fumigatus to itraconazole correlated with in vivo outcome has not been previously described. For three isolates (AF72, AF90, and AF91) of A. fumigatus from two patients with invasive aspergillosis itraconazole MICs were elevated. A neutropenic murine model was used to establish the validity of the MICs. The isolates were typed by random amplification of polymorphic DNA. Analysis of sterols, inhibition of cell-free sterol biosynthesis from [14C] mevalonate, quantitation of P-450 content, and [3H]itraconazole concentration in mycelial pellets were used to determine the mechanisms of resistance. The MICs for the three resistant isolates were >16 microg/ml. In vitro resistance was confirmed in vivo for all three isolates. Molecular typing showed the isolates from the two patients to be genetically distinct. Compared to the susceptible isolate from patient 1, AF72 had a reduced ergosterol content, greater quantities of sterol intermediates, a similar susceptibility to itraconazole in cell-free ergosterol biosynthesis, and a reduced intracellular [3H]itraconazole concentration. In contrast, AF91 and AF92 had slightly higher ergosterol and lower intermediate sterol concentrations, fivefold increased resistance in cell-free systems to the effect of itraconazole on sterol 14alpha demethylation, and intracellular [3H] itraconazole concentrations found in susceptible isolates. Resistance to itraconazole in A. fumigatus is detectable in vitro and is present in wild-type isolates, and at least two mechanisms of resistance are responsible.     

Erythrocyte membrane lateral sterol domains: a dehydroergosterol fluorescence polarization study

Structural domains of cholesterol and their regulation in the erythrocyte membrane are poorly understood. Dehydroergosterol fluorescence polarization change was used to continuously monitor the kinetics of sterol exchange and sterol domain size in erythrocyte ghost membranes. Direct correlation between molecular sterol exchange and steady-state dehydroergosterol fluorescence polarization measurements was obtained without separation of donor and acceptor membranes. Three important observations were made. First, sterol exchange between small unilamellar vesicles (SUV) with the same cholesterol/phospholipid ratio as the erythrocyte membrane (1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol = 1:1) was resolved into three kinetic cholesterol domains: 23 +/- 9% of total sterol was rapidly exchangeable, with t1/2 = 23 +/- 6 min; 59 +/- 9% of total sterol was slowly exchangeable, with t1/2 = 135 +/- 3 min; and 19 +/- 9% of total sterol was essentially nonexchangeable, with a t1/2 of days. Second, the substitution of erythrocyte ghosts for SUV as an acceptor significantly altered the kinetic parameters of sterol exchange from donor SUV, graphically showing that both the properties of the acceptor and spontaneous desorption of cholesterol from the donor SUV influenced spontaneous cholesterol transfer. Third, studies of exchange between erythrocyte ghosts revealed multiple kinetic pools of sterol differing from those in the SUV: 4 +/- 2% of total sterol was rapidly exchangeable, with t1/2 = 32 +/- 9 min; 29 +/- 3% of total sterol was very slowly exchangeable, with t1/2 = 23 +/- 7 h; and a surprisingly large 67 +/- 2% of total sterol was nonexchangeable, with a t1/2 of days.     

Cloning and characterization of ERG25, the Saccharomyces cerevisiae gene encoding C-4 sterol methyl oxidase

We have cloned the Saccharomyces cerevisiae C-4 sterol methyl oxidase ERG25 gene. The sterol methyl oxidase performs the first of three enzymic steps required to remove the two C-4 methyl groups leading to cholesterol (animal), ergosterol (fungal), and stigmasterol (plant) biosynthesis. An ergosterol auxotroph, erg25, which fails to demethylate and concomitantly accumulates 4,4-dimethylzy-mosterol, was isolated after mutagenesis. A complementing clone consisting of a 1.35-kb Dra I fragment encoded a 309-amino acid polypeptide (calculated molecular mass, 36.48 kDa). The amino acid sequence shows a C-terminal endoplasmic reticulum retrieval signal KKXX and three histidine-rich clusters found in eukaryotic membrane desaturases and in a bacterial alkane hydroxylase and xylene monooxygenase. The sterol profile of an ERG25 disruptant was consistent with the erg25 allele obtained by mutagenesis.     

Globoside as a membrane receptor: a consideration of oligosaccharide communication with the hydrophobic domain

Recognition of macromolecules by glycosphingolipids is closely correlated with the nature of the glycolipid carbohydrate; however, it is also thought to be secondarily modulated by the structure of the single fatty acid. In the present work, we sought insight into what physical effect a change in this fatty acid has on the extramembranous portion of globosides at liposomal surfaces mimicking systems for which modulated receptor function has been recorded in the past. Protons of the exocyclic hydroxymethyl group on the terminal Gal residue of globotriaosylceramide (Gb3) were replaced with deuterium. In this location, the nonperturbing probe nuclei sampled cumulative conformational and orientational characteristics of the oligosaccharide chain at a sugar residue that is critical in specific binding of verotoxins. Deuterated Gb3 having 18:1 fatty acid was compared to the same species having 22:1 fatty acid, at 6.3 mol % in unsonicated bilayers of dipalmitoylphosphatidylcholine/cholesterol. Both produced narrow, apparently axially asymmetric 2H NMR spectra over a wide temperature range. Motional properties of the terminal sugar were measurably influenced by the fluidity of the host matrix; however, evidence was not found for conformational or orientational variation in this sugar brought about by the fatty acid alteration. In related experiments, acetate protons on the terminal N-acetyl galactosamine (GalNAc) residue of globotetraosylceramide (Gb4) were substituted with deuterium, and the natural fatty acid was replaced with 18:0 or 24:0 species deuterated at C2. Once again, species with short vs long fatty acid were examined for evidence of headgroup differences. Spectra of Gb4 were compared at 10 mol % in unsonicated fluid bilayers of 1-palmitoyl-2-oleoylphosphatidylcholine, and at 5 mol % in membranes containing 33 mol% cholesterol. Spectral splittings reflecting cumulative effects on conformation and order at the terminal deuterated sugar remained unchanged between species having 18:0 vs 24:0 fatty acid in POPC/cholesterol. In a pure POPC host matrix, there was clear evidence of a motional difference between the two--the longer chain Gb4 demonstrating spectral asymmetry--but the spectral width was unchanged. Transverse relaxation times, T2, were measured. Our findings appear to help correlate the conclusions of a number of workers dealing with the molecular basis of crypticity. We suggest that changes in glycolipid receptor function based on ceramide fatty acid variation have a major origin in the fatty acid's ability to determine the thermodynamics of interaction with the host matrix, as reflected in such parameters as glycolipid motional properties, local membrane curvature, and likely glycolipid time-dependent lateral associations. The result at low concentrations of glycolipid may often be only a subtly altered collective surface epitope, best detected by a specific recognition event.     

Domains in cationic lipid plus polyelectrolyte bilayer membranes: detection and characterization via 2H nuclear magnetic resonance

2H nuclear magnetic resonance (NMR) spectroscopy of choline-deuterolabeled 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC-alpha-d2 and POPC-beta-d2) has been used to detect and quantify domain formation induced in cationic lipid-containing bilayers upon the addition of anionic polyelectrolytes. Three different polyelectrolytes, poly(sodium 4-styrenesulfonate) or PSSS, poly(sodium acrylate) or PACA, and poly(sodium glutamate) or PGLU, were added to POPC lipid bilayers containing 1,2-dioleoyl-3-(dimethylamino)propane (DODAP) as the cationic amphiphile. All three polyelectrolytes produced two-component 2H NMR spectra, consistent with two populations of POPC, one polyelectrolyte-bound and another polyelectrolyte-free. The relative intensities of the two spectral components provided the relative amounts of the two POPC populations. The 2H NMR quadrupolar splitting from either spectral component provided the DODAP content of the particular POPC population. The two POPC populations differed in that the polyelectrolyte-bound population contained a stoichiometric polyelectrolyte anion:DODAP cation ratio leading to enrichment with respect to DODAP, while the polyelectrolyte-free population was depleted of DODAP. Estimates of the size of a polyelectrolyte-defined domain revealed a constant number of bound DODAP but a flexible number of bound POPC, which increased in proportion to the global POPC content. The most compact domains were formed by the most hydrophobic polyelectrolyte, PSSS, while the most expansive domains were formed by the most hydrophilic polyelectrolyte, PGLU.     

Visual evoked potentials to illusory contours (Kanizsa''s square)

The yeast C-8,7 sterol isomerase contains a polyvalent high-affinity drug binding site similar to mammalian sigma receptors. Exogenously supplied sigma ligands inhibit sterol biosynthesis in yeast, demonstrating a pharmacological relationship between sigma ligand-binding and C-8,7 sterol isomerase activity. We report the isolation of an Arabidopsis thaliana C-8,7 sterol isomerase by functional complementation of the corresponding sterol mutant in yeast and its characterization by exposure to sigma ligands. The yeast erg2 mutant, which lacks the C-8,7 sterol isomerase gene and activity, was transformed with an Arabidopsis cDNA yeast expression library. Transformed colonies were selected for restoration of C-8,7 sterol isomerase activity (i.e. wild-type ergosterol production) by enhanced resistance to the antibiotic cycloheximide. Sterols produced in complemented lines were characterized by gas chromatography-mass spectroscopy (GC-MS). The full-length A. thaliana cDNA (pA.t.SI1) that complemented the erg2 mutation contains an open reading frame encoding a 21 kDa protein that shares 68% similarity and 35% amino acid identity to the recently isolated mouse C-8,7 sterol isomerase. The sigma ligands, haloperidol, ifenprodil and verapamil inhibited the production of ergosterol in wild-type Saccharomyces cerevisiae and in the erg2 mutant complemented with pA.t.SI1. Structural and biochemical similarities between the A. thaliana C-8,7 sterol isomerase and the mammalian emopamil-binding protein (EBP) are discussed.     

Dynamic chain conformations in dimyristoyl glycerol-dimyristoyl phosphatidylcholine mixtures. 2H-NMR studies

The dynamic molecular lipid chain conformations in fully hydrated dimyristoyl phosphatidylcholine (DMPC)-dimyristoyl glycerol (DMG) mixtures have been investigated by 2H-NMR spectroscopy of the individual lipid components, the sn-2 chains of which were perdeuterated or, in the case of DMG, specifically deuterated at the C-2 position. Mixtures of compositions corresponding to the three different regions of the binary phase diagram in which the fluid phase is lamellar (DMPC:DMG 70:30 mol/mol), inverted hexagonal (DMPC:DMG 45:55 and 40:60 mol/mol), or isotropic (DMPC:DMG 20:80 mol/mol) were investigated. The gel phase in all three regions of the phase diagram has a lamellar structure, with the lipid chains rotating about the molecular long axis but executing only limited angular excursions. In the fluid lamellar phase of the 70:30 mol/mol DMPC-DMG mixture the profile of segmental chain flexibility is similar to that in single-component phospholipid bilayers and is characterized by an order parameter plateau for both lipid components. The chain order of the DMPC component is greater than in bilayers of DMPC alone and is also greater than that of the DMG component. In the inverted hexagonal phase of the 45:55 mol/mol DMPC-DMG mixture the chain flexibility profile is characterized by more widely spaced segmental order parameters off the plateau region. The intrinsic degree of chain order in the inverted hexagonal phase is less than in the lamellar phase of the 70:30 mol/mol mixture, and the difference in chain order between the DMPC and DMG components is reduced relative to that in the lamellar phase. The unique conformational features at the C-2 position of the sn-2 chain that characterize bilayers of diacyl phospholipids are found also for the diacylglycerol molecules in the fluid lamellar phase and most probably also in the inverted hexagonal phase. The DMG molecules are therefore integrated in the membrane (or nonlamellar lipid phase) in a configuration that is similar to that of the phospholipids and different from the crystal structure of diacylglycerols.     

Spectroscopy features of the binding of polyene antibiotics to human serum albumin

The alteration in the fluorescence spectra observed for the polyene antibiotics nystatin and amphotericin B in the presence of human serum albumin is due to a decrease in the polar character of the antibiotic environment when these are bound to the protein. Amphotericin B showed two types of binding sites, the first having a very high affinity (5.8 x 10(7) M(-1)) and a secondary binding site with an affinity two orders lower than the primary site. This secondary binding site was very sensitive to temperature change. Nystatin yielded only one type of binding site with an affinity of 1.1 x 10(5) M(-1). Nystatin was found to be bound to fatty acid binding sites in albumin, while amphotericin B was not, suggesting that the fatty acid binding sites are not simple, depending on the number of unsaturated bonds on the polyene antibiotic molecule. Both polyene antibiotics displaced bilirubin bound to albumin, which is in agreement with the similarities of the affinity values of this chromophore and the polyene antibiotics with albumin.     

Investigation of membrane disruption in the reaction catalyzed by cholesterol oxidase

Dye leakage experiments were undertaken to investigate the membrane disruption properties of cholesterol oxidase. Inspection of the X-ray crystal structures of cholesterol oxidase suggested that an active-site "lid" opens in order to bind substrate [Li, J., Vrielink, A., Brick, P., & Blow, D. M. (1993) Biochemistry 32, 11507-11515]. We tested whether the interaction of the putative active-site lid with the membrane was sufficiently disruptive of the membrane structure to cause leakage or lysis of the cell membrane. Vesicles (100 nm) composed of egg phosphatidylcholine, 2-palmitoyl-3-oleoyl-1-sn-phosphatidylethanolamine, and 2-palmitoyl-3-oleoyl-1-sn-phosphatidylcholine were used in this study to mimic biomembranes. To separate the effects of membrane binding from conversion of cholesterol to cholest-4-en-3-one, the active-site mutant E361Q was utilized. In the reaction catalyzed by E361Q, isomerization of the cholest-5-en-3-one intermediate is suppressed and cholest-5-en-3-one is the major product isolated. Furthermore, E361Q produces cholest-5-en-3-one 20-fold more slowly than wild type produces cholest-4-en-3-one from cholesterol. Wild-type and E361Q cholesterol oxidases bind to vesicles with an apparent K(D) of approximately 25 microM, as measured by quenching of intrinsic tryptophan fluorescence, irrespective of headgroup size and cholesterol content. Membrane disruption was measured by leakage of the encapsulated marker carboxyfluorescein. Leakage was observed with cholesterol-containing vesicles and wild-type enzyme only; the rate of leakage was dependent on the rate of cholest-4-en-3-one production. E361Q did not induce membrane disruption, regardless of vesicle type tested. Thus, binding of cholesterol oxidase to the membrane and partitioning of cholesterol into the active site does not sufficiently perturb the bilayer to cause leakage of vesicle contents. Formation of the product cholest-4-en-3-one, however, does increase membrane permeability. Expansion of the lipid bilayer upon conversion of cholesterol to cholest-4-en-3-one is the likely cause of this increased permeability.     

Thermodynamics of partitioning of the antimalarial drug mefloquine in phospholipid bilayers and bulk solvents

The antimalarial drug mefloquine binds avidly to phospholipids in biomembranes. The thermodynamics of the partitioning process in dimyristoylphosphatidylcholine (DMPC) bilayers was investigated to give some insight into the drug-phospholipid interaction. Thermodynamic parameters for the partition equilibria were evaluated from the equilibrium partition coefficients measured as a function of temperature. Negative values of delta H and delta S were obtained for the transfer of mefloquine from the aqueous to the gel phase of the phospholipid. The partitioning is enthalpy controlled which suggests that mefloquine interacts strongly with the phospholipid phase. In contrast, the partitioning of mefloquine into the liquid crystalline phase of DMPC is entropy controlled which is typical of a hydrophobic interaction between mefloquine and the aqueous phase. The partitioning of mefloquine into the bulk solvents octanol and hexane were found to be enthalpy and entropy controlled, respectively. The enthalpy dominated partitioning of mefloquine into gel phase DMPC and octanol is attributed to the occurrence of hydrogen bonding and van der Waals interactions between solute and solvent. The flat shape of mefloquine may further aid its interaction with the orderly domains of the lipidic/organic phase. This is apparent from a comparison of the partitioning characteristics of another structurally related but conformationally different molecule, quinine into DMPC and octanol.     

Amino acid residue 149 of lecithin:cholesterol acyltransferase determines phospholipase A2 and transacylase fatty acyl specificity

Human LCAT prefers phosphatidylcholine (PC) with sn-1-palmitoyl-2-oleoyl PC (POPC) as substrate for cholesteryl ester synthesis, whereas rat LCAT (which is 92% similar in amino acid sequence) prefers sn-1-palmitoyl-2-arachidonoyl PC (PAPC). Six recombinant human LCAT cDNA clones were constructed with unique clusters of rat sequence substitutions in the human background spanning the region encoding amino acids 121-296. Media from transfected COS cells expressing each of the constructs were assayed for LCAT cholesterol esterification (CE) or phospholipase A2 (PLA2) activity using substrate particles containing POPC or PAPC. The PAPC/POPC CE activity ratio of the cluster 1 construct (amino acids 149-158) was 1.3, resembling rat LCAT, whereas cluster 2-5 clones produced CE activity ratios <0.3, unchanged from human LCAT. The cluster 6 clone (Y292H/W294F) had an intermediate ratio (0.6). Similar results were observed for LCAT PLA2 activity. In additional studies, position 149 of human LCAT was changed to the rat sequence (hE149A) and compared to a triple mutation containing the remainder of the cluster 1 changes (G151R/E154D/R158Q). CE and PLA2 activity ratio for the hE149A construct was >1.7, similar to rat LCAT, whereas the triple mutation construct retained a ratio similar to human LCAT (<0.6). Thus, a single amino acid substitution (E149A) was sufficient to alter the fatty acyl specificity of human LCAT to that of rat LCAT, with an increase in activity toward PAPC. This is the first example of a point mutation in an enzyme with PLA2 activity that results in an increase in activity toward arachidonic acid.